Costar ultra low attachment multiwell plates

Cellular viability of 100% was attributed to control wells, where cells were cultured with no discs (low control (LC) or positive growth control). Cellular viability was quantified via a colourimetric assay using invitrogen alamarBlue Cell Viability Reagent (Life Technologies Corporation, Thermo Fisher Scientific, Rockford, IL, USA). At each time point, both cell lines were exposed to alamarBlue (1:9 reagent:GM) for 1 h in the incubator (Panasonic CO₂ Incubator, MCO-170AIC, Panasonic Healthcare Co., Ltd., Tokyo, Japan) at 37° C and 5% CO₂. Then, 100 µL of supernatant was transferred into a 96-well plate in triplicates for analysis at each time point. Cell viability was measured at four-time points, day 1, 3, 5, and 10 of cell growth. The 96-well plate (Corning Costar Ultra-Low attachment multi-well plates Corning Inc., Corning, NY, USA) was then read with a UVM 340-microplate reader at excitation wavelength of 570 nm and emission wavelength of 600 nm (ASYS, Scientific laboratory supplies). Cell viability was calculated according to the following equation [35 ]: $$\mathrm{Cell} \mathrm{viability} \%=\frac{\mathrm{A}570-\left(\mathrm{A}600\times {\mathrm{R}}_0\right) \mathrm{for} \mathrm{test} \mathrm{well} }{\mathrm{A}570-\left(\mathrm{A}600\times {\mathrm{R}}_0\right) \mathrm{positive} \mathrm{growth} \mathrm{control} } \times 100$$
where A570 and A600 are absorbance at 570 and 600 nm, respectively, and R₀ is the correction factor calculated from (A570/A600) of the positive growth control.